Prokaryotic multicopy production cells, i.e. cells comprising more than one copy of a gene of interest, have been used for production of proteins of interest at industrial scale.
Preferred multicopy production cells are cells which stabile maintain the individual copies of the genes of interest during fermentation.
Further, due to environmental concerns there is an increasing desire for production cells which do not comprise any integrated antibiotic resistance genes on the chromosome and according to this line production cells which are capable of stabile maintaining the copies of the gene of interest in a fermentation medium NOT comprising an antibiotic.
EP 284126 describes a solution to the stability issue above by providing a method for constructing a prokaryotic cell comprising on the chromosome at least two copies of a gene of interest separated by endogenous DNA, which is vital (essential) to the host cell (see claim 1 of EP 284126).
The individual copies of the gene of interest are stabile maintained in a fermented cell population due to the essential DNA. If a cell crosses out this vital DNA by homologous recombination between the two copies of the gene of interest, the cell looses vital DNA and this specific cell will die.
Thereby it is possible to maintain a stable cell population comprising the copies of the gene of interest.
The method requires a knowledge of which DNA regions are vital to the cell. Alternatively, the gene is integrated on very distant places in the chromosome to have a high probability that there would be vital DNA between the copies (see FIG. 2 of EP 284126). This is a relatively laborious process and uncertain process.